State Key Laboratory of Military Stomatology, National Clinical Research Center for Oral Diseases, Shaanxi Clinical Research Center for Oral Diseases, Fourth Military Medical University, Xi'an, 710032, China.
Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi'an, 710032, China.
J Orthop Surg Res. 2022 Jul 6;17(1):338. doi: 10.1186/s13018-022-03230-1.
ClC-3 chloride channels promote osteogenic differentiation. Transforming growth factor-β1 (TGF-β1) and its receptors are closely related to ClC-3 chloride channels, and canonical TGF-β1 signaling is largely mediated by Smad proteins. The current study aimed to explore the role of the Smad2/3/4 signaling pathway in the mechanism by which ClC-3 chloride channels regulate osteogenic differentiation in osteoblasts.
First, real-time PCR and western blotting were used to detect the expression of Smad and mitogen-activated protein kinase (MAPK) proteins in response to ClC-3 chloride channels. Second, immunocytochemistry, coimmunoprecipitation (Co-IP) and immunofluorescence analyses were conducted to assess formation of the Smad2/3/4 complex and its translocation to the nucleus. Finally, markers of osteogenic differentiation were determined by real-time PCR, western blotting, ALP assays and Alizarin Red S staining.
ClC-3 chloride channels knockdown led to increased expression of Smad2/3 but no significant change in p38 or Erk1/2. Furthermore, ClC-3 chloride channels knockdown resulted in increases in the formation of the Smad2/3/4 complex and its translocation to the nucleus. In contrast, the inhibition of TGF-β1 receptors decreased the expression of Smad2, Smad3, p38, and Erk1/2 and the formation of the Smad2/3/4 complex. Finally, the expression of osteogenesis-related markers were decreased upon ClC-3 and Smad2/3/4 knockdown, but the degree to which these parameters were altered was decreased upon the knockdown of ClC-3 and Smad2/3/4 together compared to independent knockdown of ClC-3 or Smad2/3/4.
The Smad2/3 proteins respond to changes in ClC-3 chloride channels. The Smad2/3/4 signaling pathway inhibits osteogenic differentiation regulation by ClC-3 chloride channels in MC3T3-E1 cells.
ClC-3 氯离子通道促进成骨细胞分化。转化生长因子-β1（TGF-β1）及其受体与 ClC-3 氯离子通道密切相关，经典的 TGF-β1 信号主要由 Smad 蛋白介导。本研究旨在探讨 Smad2/3/4 信号通路在 ClC-3 氯离子通道调节成骨细胞成骨分化中的作用机制。
首先，通过实时 PCR 和 Western blot 检测 ClC-3 氯离子通道对 Smad 和丝裂原活化蛋白激酶（MAPK）蛋白表达的影响。其次，通过免疫细胞化学、免疫共沉淀（Co-IP）和免疫荧光分析评估 Smad2/3/4 复合物的形成及其向核内的转位。最后，通过实时 PCR、Western blot、ALP 测定和茜素红 S 染色检测成骨分化标志物。
ClC-3 氯离子通道敲低导致 Smad2/3 表达增加，但 p38 或 Erk1/2 无明显变化。此外，ClC-3 氯离子通道敲低导致 Smad2/3/4 复合物形成增加并向核内转位。相反，TGF-β1 受体抑制剂降低了 Smad2、Smad3、p38 和 Erk1/2 的表达以及 Smad2/3/4 复合物的形成。最后，ClC-3 和 Smad2/3/4 敲低后成骨相关标志物的表达减少，但与单独敲低 ClC-3 或 Smad2/3/4 相比，ClC-3 和 Smad2/3/4 同时敲低时这些参数的变化程度降低。
Smad2/3 蛋白对 ClC-3 氯离子通道的变化作出反应。Smad2/3/4 信号通路抑制 ClC-3 氯离子通道对 MC3T3-E1 细胞成骨分化的调节。
